The present invention relatesto a diisopropyl-fluorophosphatase (enzyme classification EC 3.1.8.2., called DFPase hereinafter) from Loligo vulgaris, to the base sequence encoding the enzyme, and to the use of the enzyme and a method for its production in transformed cells.
The term DFPase has a wide scope in the description of a class of enzymes able to hydrolyse diisopropyl fluorophosphate (DFP) and other organophosphorus compounds with a similar structure. The group of these organophosphorus compounds can be traced back to compounds of the following basic structure: 
In this, Z represents oxygen or sulphur, Y represents a group which may, as Hxe2x80x94Y compound, have acidic properties. These are, inter alia, anhydride groups (deprotonated acid groups)(in particular an F or CN group) or ester groups such as a thioester, an enol ester or a p-nitrophenyl ester group. The groups X1 and X2 may be straight- or branched-chain or cyclic alkoxy, alkyl, aryl, alkylamino or dialkylamino groups containing 1 to 15 carbon atoms. A large number of such compounds are or were used as insecticides. Other compounds are covered by the term of so-called nerve gases. The nerve gases of this class of compounds include, inter alia, DFP, tabun, soman, sarin, thylsarin and cyclosarin.
Reducing the stocks of these highly toxic compounds in the world is an increasing problem. In addition, it is necessary to find environmentally acceptable methods for decontaminating already contaminated areas of the environment.
The most important method to date for destroying large amounts of these substances comprises incineration at high temperatures.
In addition, for example, U.S. Pat. No. 4,666,696 deals with the destruction of nerve gases and other cholinesterase inhibitors by reduction with molten aluminium.
Further approaches to solving this problem comprise, for example, the use of physical methods. Thus, for example, U.S. Pat. No. 5,550,311 describes the thermal decomposition of toxic compounds (nerve gases) in an aqueous stream. Another approach is described in U.S. Pat. No. 5,648,591, in which chemical weapons such as, for example, sarin are broken down by activation in mechanical mills. All these methods have severe disadvantages in relation to their economic efficiency and their limited range of applications. Thus, for example, it is necessary in all these methods to feed the appropriate toxic substances into particular apparatuses, which makes, for example, use in the field difficult if not impossible.
Biological disposal of chemical weapons stocks and decontamination by microorganisms or enzymes which can be produced in large quantities might provide a simplification or a more effective approach to solving these problems.
The present invention therefore contrasts with the decontamination methods described above and builds on investigations by Aldrige as long ago as 1953. Aldridge observed that various tissues from different organisms contain enzymes which hydrolyse paraoxon, a cholinesterase inhibitor. Later investigations in 1966 by Hoskin and co-workers showed that the squid Loligo pealii contains a DFP-cleaving enzyme. Numerous investigations in this area have followed to the present day, but it has not yet been possible to sequence or even isolate a recombinant protein. The enzyme investigated by Hoskin could not be accurately characterized in terms of its molecular weight. Whereas Hoskin assumed a molecular weight of 26,600 Da, other research groups, for example Kopec-Smyth et al. (1993), regarded it as probable that the 26.6 kDa protein is only a fragment of a very unstable 42 kDa protein. A short amino acid sequence of a peptide fragment of this DFPase was published in 1993 by Ward and Deschamps. The sequence section described therein is not found in the claimed amino acid sequence of the present invention, for which reason it must be assumed that the DFPase from Loligo pealii differs distinctly from the DFPase of the present invention.
Similar proteins have been identified in a wide variety of organisms apart from Loligo pealii. Thus, they have been found inter alia in thermophilic or halophilic bacteria, in E. coli, Proteus vulgaris, Saccharomyces cerevisiae, Pseudomonas diminuta, Flavobacterium and the eukaryotic unicellular organism Tetrahymena thermophilia, and in insects and invertebrates. Detection has also been possible in mammals such as mice, rats, rabbits, pigs and humans. Despite the efforts of a large number of research groups concerned with enzymes which resemble in their specificity the DFPase from Loligo vulgaris of the present invention, none of these groups has succeeded incompletely elucidating the amino acid sequence, and the nucleotide sequence underlying the latter, of an enzyme corresponding to the present invention, in transforming the corresponding nucleotide sequence using a vector into a host cell, and thus in making industrial production possible in excellent purity. Although Dierl (1995, thesis, Johann Wolfgang Goethe University, Frankfurt/Main) was able during his research work to elucidate a partial sequence of the DFPase from Loligo vulgaris, no details of the amino acid sequence or nucleotide sequence are to be found in this study. The partial information provided by Dierl is incomplete and reproducibility of the results presented therein is impossible simply because of the unavailability to the public of the cDNA gene bank on which the study is based but which is not accessible to the public.
One of the objects of the present invention is to provide the complete and functioning DFP-cleaving enzyme from Loligo vulgaris and the nucleotide sequence encoding the enzyme in order to make industrial production of a genetic engineering type possible. A further object was to provide a DFPase which can be isolated on an industrial scale without loss of stability by fractional ammonium sulphate precipitation and which has its activity optimum at a neutral pH of about 7.5 and room temperature (about 25xc2x0 C.). It was intended thereby not least to achieve the object of environmentally friendly, energy-saving disposal of nerve gases and insecticides. A further object to be achieved was to provide a storage- and solvent-stable DFPase. Thus, it was intended, for example, that a concentrated solution be stable with negligible loss of activity at 4xc2x0 C. for a lengthy period. It was also intended that various solvents or solvent-containing aqueous media (for example 10% strength aqueous ethanolic solution) have no effect on the activity of the enzyme. A further object to be achieved was to provide a DFPase which is active in a wide variety of buffer systems. The provision of the DFPase was further intended to make it possible to decontaminate, detoxify and moreover detect acetylcholinesterase inhibitors which serve as substrate of the DFPase. The provision of such an enzyme is intended not only to make the industrial destruction of appropriate nerve gases or insecticides possible but also to provide the possibility of decontaminating contaminated habitats (soils, watercourses, etc.). A cloning in plants is also possible thereby. It is further conceivable to employ enzyme for producing medicinal products for detoxifying or treating humans and animals. This might involve the enzyme being employed, for example, locally in the form of a skin cream, parenterally in the form of a solution for infusion or inhalation, or else orally.
The said objects have been achieved by providing a DFPase of the following amino acid sequence (SEQ ID NO: 1):
The claimed diisopropyl-fluorophosphatase also embraces in this connection amino acid sequences which can be derived from the above sequence by a deletion, insertion and/or substitution of one or more amino acids, as long as the DFPase activity is retained. This also includes, of course, a truncation of the amino-and/or carboxy-terminal side.
This amino acid sequence according to the invention has as its basis a DNA sequence according to the invention which comprises a DNA sequence which codes for the DFPase from Loligo vulgaris and which comprises in a preferred embodiment the following nucleotide sequence (SEQ ID NO: 2).
It is possible by modifications of the nucleotide sequence to adapt, for example, the substrate specificity, solubility and/or stability of the enzyme to the problems arising.
A further object of the present invention is to provide a vector which contains at least one copy of a DNA sequence according to the invention. This vector may be any prokaryotic or eukaryotic vector on which the DNA sequence according to the invention is present, preferably under the control of an expression signal. Expression signals include, inter alia, promoters, operators and enhancers. Promoters may be, for example, a T7 promoter or lac, tac, lacUV5 or PL promoters. Suitable prokaryotic vectors are, for example, chromosomal vectors such as, for example bacteriophages, (for example, bacteriophage xcex) and extrachromosomal vectors such as, for example, plasmids, with circular plasmid vectors being preferred in this connection. The vector according to the invention may, on the other hand, also be a eukaryotic vector, for example a yeast vector or a vector suitable for higher cells, such as, for example, a plasmid vector or a viral vector. Vectors of the indicated types are familiar to the person skilled in the area of molecular biology so that no further details on this are necessary here. It is possible in a preferred embodiment to use as such vectors so-called secretion vectors which make it possible to express a protein which is a fusion between DFPase and a signal peptide, where the signal peptide is responsible for passing the protein through the cell membrane and being separated accurately from the DFPase by signal peptidases on secretion. Suitable signal peptides are, for example, a pelB, xcex1 or PHO1 signal peptide. The linkage of the DFPase to such signal peptides is advantageous in order to prevent the formation of inclusion bodies, that is to say DFPase aggregates within the cell, and to pass larger amounts of protein into the periplasmic space or the medium.
The present invention further relates to a cell which is transformed with a DNA sequence according to the invention or a vector according to the invention. This cell can be, for example, a prokaryotic cell, preferably a Gram-negative prokaryotic cell, particular preferably a eubacterial cell. In one embodiment, this cell is an E. coli cell. The transformation of prokaryotic cells with exogenous nucleic acid sequences is familiar to the person skilled in the area of molecular biology. The cell according to the invention may, however, also be a eukaryotic cell such as, for example, a fungus cell (for example yeast), an animal or plant cell. Preferred eukaryotic expression systems are, for example, Pichia pastoris or Saccharomyces cerevisiae. The transformation or transfection of eukaryotic cells with exogenous nucleic acid sequences is familiar to a person skilled in the area of molecular biology and therefore needs no further explanation.
The provision of the abovementioned proteins, DNA sequences, vectors and transformed cells makes the industrial production of an enzyme with DFPase activity possible. This makes it possible for the first time to degrade, in a simple, economic and environmentally compatible manner, large quantities of acetylcholinesterase inhibitors.
This may entail the DFPase being obtained by recombinant DNA technology as a constituent of an extract from the host organism or in isolated and purified form, for example by expression in E. coli. The use according to the invention of such a DFPase comprises the degradation of Pxe2x80x94F linkages or of acetylcholinesterase inhibitors corresponding to the basic formula and containing Pxe2x80x94Y linkages. It is possible and preferred for this purpose to employ the purified and isolated DFPase for example for the industrial cleavage of acetylcholinesterase inhibitors containing Pxe2x80x94Y linkages (corresponding to the basic formula) in an enzyme reactor. The immobilization of enzymes in such a reactor is familiar to the skilled person and therefore need not be described in detail here. It is conceivable inter alia to polymerize the DFPase into foams such as, for example, polyurethane foams. Another embodiment of the use is, for example, the use of the DFPase in an intact microorganism which can be employed, for example, for decontaminating soils outdoors, and transformed soil bacteria are preferably employed in this case. A further use according to the invention comprises employing a DFPase according to the invention in a foam, where the foam acts as carrier and/or wetting substance. A foam of this type may be a surfactant foam which can be employed inter alia for decontaminating soils, surfaces, valuable equipment or the like. In some cases, a simple spraying, that is to say application as aerosol, may also suffice. The use in foam form is then not necessarily obligatory. It is also conceivable to use the DFPase in methods operating in a stationary manner and making use, for example, of reactors, and to use the DFPase in a mobile manner for the decontamination of equipment or large areas outdoors. Further embodiments according to the invention of the use of the DFPase may therefore also comprise the detoxification of contaminated watercourses or of drinking water. Immobilization of the enzyme by use thereof as stationary phase in a reactor can take place, for example, by covalent linkage of the enzyme to a solid carrier.
The coding cDNA may have a xe2x80x9cHis tagxe2x80x9d attached, which makes it possible to produce a modified DFPase. This modified DFPase is able to bind to carrier material modified with nickel NTA. If the carrier material modified with nickel NTA is the packing material for a separating column, this method can be used for purifying the DFPase.
It is furthermore possible to impregnate textiles with the enzyme by covalent or noncovalent linkage in order to serve as protective clothing.
One use according to the invention may also consist of novel methods of detection, for example biosensors. In this case, the DFPase acts as receptor which is immobilized on a transducer. If the DFPase cleaves the analyte to be detected, that is to say the substrates described above, the biological signal is transformed into a corresponding measurable electrical signal which is amplified by an electronic component. The final signal is generally related to the quantity and/or nature of the analyte. The DFPase can in this case be present as receptor in pure isolated form or in the cell expressing it. Suitable transducers are the transformer components known to the person skilled in the area of biosensors.
A further use according to the invention is the provision of medicinal products which contain the DFPase as active ingredient and are thus able to contribute to detoxifying or treating a human or animal poisoned with an acetylcholinesterase inhibitor containing Pxe2x80x94Y linkages. Suitable for this besides local administration is also parenteral or oral administration.
The present invention further relates to a method for producing a DFPase in which the latter is produced by the cell according to the invention. This method thus contrasts with a complete chemical synthesis of the DFPase. In the method according to the invention for producing a DFPase by a transformed cell, the transformation with an expression vector is preceded by the following steps,
(1) isolation of the mRNA of Loligo vulgaris from the head ganglia thereof which have been removed immediately after sacrifice and which should be immediately deep-frozen in liquid nitrogen,
(2) purification of the mRNA by affinity chromatography on an oligo-(dR)-cellulose column,
(3) translation of the mRNA into cDNA.
(4) cloning of the cDNA into xcex phages,
(5) finding the xcex phages containing the genetic information of the DFPase by means of degenerate probes using the polymerase chain reaction,
(6) amplification of the genetic information by means of the polymerase chain reaction,
(7) transferring the genetic information into an expression vector.
In this method it was necessary to overcome various difficulties which the person skilled in the area of genetic engineering could not have solved with the standard techniques available to him. Thus, for example, it was not obvious, but was absolutely necessary, to isolate the mRNA from Loligo vulgaris from the head ganglia of a freshly sacrificed animal, it being necessary to store the head ganglia in liquid nitrogen immediately after their removal. If this procedure is not followed, the chances of isolating a full length mRNA of the DFPase are virtually zero. The small number of DFPase-encoding mRNA molecules moreover makes it absolutely necessary to use the polymerase chain reaction technique. To find the genetic information in the cDNA bank it was furthermore necessary to develop highly specific probes which had to agree in long regions with the cDNA from Loligo vulgaris. Synthesis of these probes was furthermore impeded by the fact that, at the time of the invention, no relatively long protein sequences of the DFPase from Loligo vulgaris which would have made it possible to synthesize highly specific probes were known. The person skilled in the area of molecular biology is able to synthesize such probes in a simple manner only through the sequence information provided in the present invention.
The following example is intended to illustrate the invention.